ABSTRACT
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein. This epithelial anion channel regulates the active transport of chloride and bicarbonate ions across membranes. Mutations result in reduced surface expression of CFTR channels with impaired functionality. Correctors are small molecules that support the trafficking of CFTR to increase its membrane expression. Such correctors can have different mechanisms of action. Combinations may result in a further improved therapeutic benefit. We describe the identification and optimization of a new pyrazolol3,4-bl pyridine-6-carboxylic acid series with high potency and efficacy in rescuing CFTR from the cell surface. Investigations showed that carboxylic acid group replacement with acylsulfonamides and acylsulfonylureas improved ADMET and PK properties, leading to the discovery of the structurally novel co-corrector GLPG2737. The addition of GLPG2737 to the combination of the potentiator GLPG1837 and C1 corrector 4 led to an 8-fold increase in the F508del CFTR activity.
Subject(s)
Cystic Fibrosis , Humans , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Mutation , Cell Membrane/metabolism , Carboxylic Acids/therapeutic use , Benzodioxoles/pharmacology , Aminopyridines/therapeutic useABSTRACT
Salt-inducible kinases (SIKs) SIK1, SIK2, and SIK3 are serine/threonine kinases and form a subfamily of the protein kinase AMP-activated protein kinase (AMPK) family. Inhibition of SIKs in stimulated innate immune cells and mouse models has been associated with a dual mechanism of action consisting of a reduction of pro-inflammatory cytokines and an increase of immunoregulatory cytokine production, suggesting a therapeutic potential for inflammatory diseases. Following a high-throughput screening campaign, subsequent hit to lead optimization through synthesis, structure-activity relationship, kinome selectivity, and pharmacokinetic investigations led to the discovery of clinical candidate GLPG3312 (compound 28), a potent and selective pan-SIK inhibitor (IC50: 2.0 nM for SIK1, 0.7 nM for SIK2, and 0.6 nM for SIK3). Characterization of the first human SIK3 crystal structure provided an understanding of the binding mode and kinome selectivity of the chemical series. GLPG3312 demonstrated both anti-inflammatory and immunoregulatory activities in vitro in human primary myeloid cells and in vivo in mouse models.
Subject(s)
AMP-Activated Protein Kinases , Protein Serine-Threonine Kinases , Mice , Animals , Humans , Gene Expression , CytokinesABSTRACT
There are currently no approved disease-modifying osteoarthritis (OA) drugs (DMOADs). The aggrecanase ADAMTS-5 is key in the degradation of human aggrecan (AGC), a component of cartilage. Therefore, ADAMTS-5 is a promising target for the identification of DMOADs. We describe the discovery of GLPG1972/S201086, a potent and selective ADAMTS-5 inhibitor obtained by optimization of a promising hydantoin series following an HTS. Biochemical activity against rat and human ADAMTS-5 was assessed via a fluorescence-based assay. ADAMTS-5 inhibitory activity was confirmed with human aggrecan using an AGC ELISA. The most promising compounds were selected based on reduction of glycosaminoglycan release after interleukin-1 stimulation in mouse cartilage explants and led to the discovery of GLPG1972/S201086. The anticatabolic activity was confirmed in mouse cartilage explants (IC50 < 1.5 µM). The cocrystal structure of GLPG1972/S201086 with human recombinant ADAMTS-5 is discussed. GLPG1972/S201086 has been investigated in a phase 2 clinical study in patients with knee OA (NCT03595618).
Subject(s)
ADAMTS5 Protein/antagonists & inhibitors , Osteoarthritis/drug therapy , ADAMTS5 Protein/metabolism , Animals , Cartilage, Articular/drug effects , Cartilage, Articular/metabolism , Dogs , Glycosaminoglycans/metabolism , Humans , Mice , Mice, Inbred C57BL , Models, Molecular , Osteoarthritis/metabolism , RatsABSTRACT
GPR84 is a medium chain free fatty acid-binding G-protein-coupled receptor associated with inflammatory and fibrotic diseases. As the only reported antagonist of GPR84 (PBI-4050) that displays relatively low potency and selectivity, a clear need exists for an improved modulator. Structural optimization of GPR84 antagonist hit 1, identified through high-throughput screening, led to the identification of potent and selective GPR84 inhibitor GLPG1205 (36). Compared with the initial hit, 36 showed improved potency in a guanosine 5'-O-[γ-thio]triphosphate assay, exhibited metabolic stability, and lacked activity against phosphodiesterase-4. This novel pharmacological tool allowed investigation of the therapeutic potential of GPR84 inhibition. At once-daily doses of 3 and 10 mg/kg, GLPG1205 reduced disease activity index score and neutrophil infiltration in a mouse dextran sodium sulfate-induced chronic inflammatory bowel disease model, with efficacy similar to positive-control compound sulfasalazine. The drug discovery steps leading to GLPG1205 identification, currently under phase II clinical investigation, are described herein.
Subject(s)
Drug Discovery/methods , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/metabolism , Acetates/chemistry , Acetates/pharmacology , Allosteric Regulation/drug effects , Allosteric Regulation/physiology , Animals , Caco-2 Cells , Cells, Cultured , Dogs , Drug Evaluation, Preclinical/methods , Female , HEK293 Cells , Humans , Male , Mice , Mice, Inbred BALB C , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
Medium chain fatty acids can activate the pro-inflammatory receptor GPR84 but so also can molecules related to 3,3'-diindolylmethane. 3,3'-Diindolylmethane and decanoic acid acted as strong positive allosteric modulators of the function of each other and analysis showed the affinity of 3,3'-diindolylmethane to be at least 100 fold higher. Methyl decanoate was not an agonist at GPR84. This implies a key role in binding for the carboxylic acid of the fatty acid. Via homology modelling we predicted and confirmed an integral role of arginine172, located in the 2nd extracellular loop, in the action of decanoic acid but not of 3,3'-diindolylmethane. Exemplars from a patented series of GPR84 antagonists were able to block agonist actions of both decanoic acid and 3,3'-diindolylmethane at GPR84. However, although a radiolabelled form of a related antagonist, [3H]G9543, was able to bind with high affinity to GPR84, this was not competed for by increasing concentrations of either decanoic acid or 3,3'-diindolylmethane and was not affected adversely by mutation of arginine172. These studies identify three separable ligand binding sites within GPR84 and suggest that if medium chain fatty acids are true endogenous regulators then co-binding with a positive allosteric modulator would greatly enhance their function in physiological settings.
Subject(s)
Receptors, Cell Surface/metabolism , Binding Sites , Decanoic Acids/metabolism , Humans , Indoles/metabolism , Ligands , Receptors, G-Protein-Coupled , Structure-Activity RelationshipABSTRACT
Through their function as epigenetic readers of the histone code, the BET family of bromodomain-containing proteins regulate expression of multiple genes of therapeutic relevance, including those involved in tumor cell growth and inflammation. BET bromodomain inhibitors have profound antiproliferative and anti-inflammatory effects which translate into efficacy in oncology and inflammation models, and the first compounds have now progressed into clinical trials. The exciting biology of the BETs has led to great interest in the discovery of novel inhibitor classes. Here we describe the identification of a novel tetrahydroquinoline series through up-regulation of apolipoprotein A1 and the optimization into potent compounds active in murine models of septic shock and neuroblastoma. At the molecular level, these effects are produced by inhibition of BET bromodomains. X-ray crystallography reveals the interactions explaining the structure-activity relationships of binding. The resulting lead molecule, I-BET726, represents a new, potent, and selective class of tetrahydroquinoline-based BET inhibitors.
Subject(s)
Aminoquinolines/chemical synthesis , Anti-Inflammatory Agents/chemical synthesis , Apolipoprotein A-I/metabolism , Benzoates/chemical synthesis , Nuclear Proteins/antagonists & inhibitors , Protein Serine-Threonine Kinases/antagonists & inhibitors , Quinolines/chemical synthesis , Transcription Factors/antagonists & inhibitors , Aminoquinolines/pharmacokinetics , Aminoquinolines/pharmacology , Animals , Anti-Inflammatory Agents/pharmacokinetics , Anti-Inflammatory Agents/pharmacology , Benzoates/pharmacokinetics , Benzoates/pharmacology , Cell Cycle Proteins , Drug Discovery , Humans , Mice , Quinolines/pharmacokinetics , Quinolines/pharmacology , Structure-Activity RelationshipABSTRACT
The bromo and extra C-terminal domain (BET) family of bromodomains are involved in binding epigenetic marks on histone proteins, more specifically acetylated lysine residues. This paper describes the discovery and structure-activity relationships (SAR) of potent benzodiazepine inhibitors that disrupt the function of the BET family of bromodomains (BRD2, BRD3, and BRD4). This work has yielded a potent, selective compound I-BET762 that is now under evaluation in a phase I/II clinical trial for nuclear protein in testis (NUT) midline carcinoma and other cancers.
Subject(s)
Antineoplastic Agents/pharmacology , Benzodiazepines/pharmacology , Nuclear Proteins/antagonists & inhibitors , Transcription Factors/antagonists & inhibitors , Animals , Anti-Inflammatory Agents/chemical synthesis , Anti-Inflammatory Agents/pharmacokinetics , Anti-Inflammatory Agents/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacokinetics , Apolipoprotein A-I/biosynthesis , Benzodiazepines/chemical synthesis , Benzodiazepines/pharmacokinetics , Cell Cycle Proteins , Dogs , Epigenesis, Genetic , Humans , Macaca fascicularis , Mice , Models, Molecular , Permeability , Protein Structure, Tertiary , Rats , Stereoisomerism , Structure-Activity RelationshipABSTRACT
BET family proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Selective inhibitors of BET proteins exhibit potent anti-proliferative activity in a number of hematologic cancer models, in part through suppression of the MYC oncogene and downstream Myc-driven pathways. However, little is currently known about the activity of BET inhibitors in solid tumor models, and whether down-regulation of MYC family genes contributes to sensitivity. Here we provide evidence for potent BET inhibitor activity in neuroblastoma, a pediatric solid tumor associated with a high frequency of MYCN amplifications. We treated a panel of neuroblastoma cell lines with a novel small molecule inhibitor of BET proteins, GSK1324726A (I-BET726), and observed potent growth inhibition and cytotoxicity in most cell lines irrespective of MYCN copy number or expression level. Gene expression analyses in neuroblastoma cell lines suggest a role of BET inhibition in apoptosis, signaling, and N-Myc-driven pathways, including the direct suppression of BCL2 and MYCN. Reversal of MYCN or BCL2 suppression reduces the potency of I-BET726-induced cytotoxicity in a cell line-specific manner; however, neither factor fully accounts for I-BET726 sensitivity. Oral administration of I-BET726 to mouse xenograft models of human neuroblastoma results in tumor growth inhibition and down-regulation MYCN and BCL2 expression, suggesting a potential role for these genes in tumor growth. Taken together, our data highlight the potential of BET inhibitors as novel therapeutics for neuroblastoma, and suggest that sensitivity is driven by pleiotropic effects on cell growth and apoptotic pathways in a context-specific manner.
Subject(s)
Benzodiazepines/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Gene Silencing , Neuroblastoma/genetics , Neuroblastoma/metabolism , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Oncogene Proteins/genetics , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/genetics , RNA-Binding Proteins/antagonists & inhibitors , Transcription Factors/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Antineoplastic Agents/toxicity , Apoptosis/drug effects , Apoptosis/genetics , Benzodiazepines/chemistry , Benzodiazepines/toxicity , Cell Cycle Proteins , Cell Proliferation/drug effects , Cluster Analysis , Disease Models, Animal , Female , Gene Expression Profiling , Gene Regulatory Networks , Humans , Kinetics , Mice , Models, Molecular , Molecular Conformation , N-Myc Proto-Oncogene Protein , Neuroblastoma/drug therapy , Neuroblastoma/pathology , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Protein Binding , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Signal Transduction/drug effects , Transcription Factors/chemistry , Transcription Factors/metabolism , Tumor Burden/drug effects , Tumor Burden/genetics , Xenograft Model Antitumor AssaysABSTRACT
The discovery, synthesis and biological evaluation of a novel series of 7-isoxazoloquinolines is described. Several analogs are shown to increase ApoA1 expression within the nanomolar range in the human hepatic cell line HepG2.
Subject(s)
Apolipoprotein A-I/metabolism , Drug Discovery , Heterocyclic Compounds, 4 or More Rings/chemistry , Heterocyclic Compounds, 4 or More Rings/pharmacology , Nuclear Proteins/antagonists & inhibitors , Quinolines/chemistry , Up-Regulation/drug effects , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Hep G2 Cells , Histone Acetyltransferases , Histone Chaperones , Humans , Inhibitory Concentration 50 , Mice , Mice, Inbred BALB C , Nerve Tissue Proteins , Nuclear Proteins/metabolism , Quinolines/pharmacology , Rats , Structure-Activity RelationshipABSTRACT
Epigenetic mechanisms of gene regulation have a profound role in normal development and disease processes. An integral part of this mechanism occurs through lysine acetylation of histone tails which are recognized by bromodomains. While the biological and structural characterization of many bromodomain containing proteins has advanced considerably, the therapeutic tractability of this protein family is only now becoming understood. This paper describes the discovery and molecular characterization of potent (nM) small molecule inhibitors that disrupt the function of the BET family of bromodomains (Brd2, Brd3, and Brd4). By using a combination of phenotypic screening, chemoproteomics, and biophysical studies, we have discovered that the protein-protein interactions between bromodomains and acetylated histones can be antagonized by selective small molecules that bind at the acetylated lysine recognition pocket. X-ray crystal structures of compounds bound into bromodomains of Brd2 and Brd4 elucidate the molecular interactions of binding and explain the precisely defined stereochemistry required for activity.